大鼠nelin基因干扰质粒的构建及其体外抑制效果研究  

The Construction and in vitro Study of Inhibition Effect of shRNA Plasmid targeting rat nelin

作　　者：王慧[1] 王林[1] 宋莉[1] 叶珏[1] 孟宪敏[1] 

机构地区：[1]北京协和医学院中国医学科学院阜外心血管病医院阜外心血管病研究所中心实验室,北京100037

出　　处：《中国分子心脏病学杂志》2010年第1期4-10,共7页Molecular Cardiology of China

基　　金：国家自然科学基金自助项目(30871057)

摘　　要：目的构建大鼠nelin基因干扰质粒并对其进行体外抑制效果验证,为研究nelin在心脏发育、心肌肥厚、心衰及其它心脏疾病中的作用提供有效的研究工具。方法提取大鼠心肌组织RNA,逆转录为cDNA,设计合成含BamHI和EcoRI酶切位点的引物,以大鼠cDNA为模板PCR扩增获得nelin基因,定向克隆入pEGFP-N1-3FLAG载体,测序验证。以克隆得到的大鼠nelin基因为模板,设计合成候选干扰序列,亚克隆至pGCSIL-U6载体,获得干扰质粒。以nelin基因干扰质粒和重组表达质粒共转染HEK293T细胞,WesternBlot方法检测干扰效果。结果成功克隆了大鼠nelin基因并构建了nelin基因重组表达载体pEGFP-N1-3FLAG-rNelin。设计构建的4个干扰质粒(靶位点分别为+370-388位,+376-394位,+545-563位,[phone],命名为pGCSIL-U6-nelin/siteⅠ,Ⅱ,Ⅲ,Ⅳ)均可显著降低nelin基因的表达,尤以靶向(+370-388位)位点即pGCSIL-U6/nelin-siteI质粒的干扰效果最佳。结论本实验构建的大鼠nelin基因特异性干扰质粒能从蛋白水平上高效特异地抑制ne-lin基因表达,为进一步开展其功能研究奠定了基础。Objective To provide a useful tool for studying the function of nelin during the heart development,hypertrophy,heart failure and other heart diseases,the RNA-interference plasmids targeting rat nelin gene were constructed,of which silencing effect were evaluated in this study.Methods A pair of primers based on human nelin gene with BamH I and EcoR I restriction site were designed and synthesized.Fragment was amplified with cDNA template reversed from rat heart RNA and cloned into the pEGFP-N1-3FLAG vector,which was identified by sequencing.Four specific interference sequences targeting rat nelin （the target sites are ＋370-388,＋376-394,＋545-563 and [phone]） were designed,synthesized and inserted into the pGCSIL-U6 vector respectively.RNA interference plasmids （pGCSIL-U6-nelin/site Ⅰ,Ⅱ,Ⅲ,Ⅳ） and recombinant expression plasmids （pEGFP-N1-3FLAG-rNelin） were co-transfected into HEK293T cells.The expression of Nelin-FLAG was detected by western blot.Results pEGFP-N1-3FLAG-rNelin was constructed successfully.All of the RNA interference plasmids constructed in this study could reduce the expression of rat Nelin in 293T cell line,especially the pGCSIL-U6-nelin/site I had the ideal distinct effect.Conclusion Rat nelin-specific RNA interference plasmids constructed in this research,as an effective tool for further function study of Nelin,can knock down the expression of rat nelin efficiently in protein level.

关 键 词：nelin基因 RNA干扰 SHRNA 载体构建 重组表达质粒 体外抑制 大鼠 设计合成 酶切位点 基因表达 

分 类 号：Q78[生物学—分子生物学]